1. Field of the Invention
The interest in analyzing for trace amounts of a wide variety of organic materials, such as drugs, contaminants, pollutants, and the like, has engendered efforts to provide simpler and more accurate techniques for measuring low concentrations of compounds of interest. One group of techniques is referred to as immunoassays, which are based on the ability to have a compound, usually an antibody, which is capable of recognizing or specifically binding to a compound having a specific spatial and polar organization. The specific binding pair may be referred to as ligand and receptor (antiligand).
In performing the immunoassays, normally the ligand is labeled with a label which provides a detectible signal and the labeled ligand is allowed to compete with the ligand in the sample for a limited amount of the antiligand. The immunoassay techniques then provide for distinguishing between the associated labeled ligand and antiligand and dissociated labeled ligand which is free in the bulk solution. Distinguishing the associated signal label may be achieved by separating the unassociated signal label from the associated signal label and determining the amount of signal label in either of the fractions.
A preferred method is to employ a procedure which does not require separation; one distinguishes associated signal label from unassociated signal label by there being a substantial difference in the level of signal between the two. One of the problems with the latter technique is the fact that the signal label which is measured is not freed from materials present in the assay system which may provide a background or cause non-specific interference with the signal measurement.
For many ligands of interest, particularly large molecules, such as proteins, polysaccharides, nucleic acids, and the like, obtaining the ligand in pure form is frequently difficult, and in some instances impossible. Furthermore, where the antiligand is an antibody, the antibody is normally isolated as a complex mixture of globulins, of which a portion, usually less than 50%, is the antibody of interest. Where one is labeling the impure ligand or antiligand, a substantial proportion of the label will be conjugated to molecules other than the ligand or antiligand. These labels will be capable of providing a detectible signal, which will act as a background for the measurement. That is, these labels will provide a base value which will be additive to the value obtained from the label bound to the ligand or antiligand. Where one is determining a small value between two large values, substantial errors and uncertainities are introduced. For example, where there are non-specific effects affecting the label, the presence of a large amount of label unrelated to ligand or antiligand will greatly increase the variability due to the non-specific effects on a sample-by-sample basis.
It is therefore desirable to provide techniques which will allow for discrimination between label bound to ligand or antiligand participating in the assay and related to the amount of analyte in the assay and label which is present which is not involved with ligand or antiligand. The techniques provided must, therefore, be able to discriminate between the label which is providing signal related to the amount of analyte in the medium and the signal being obtained from label unrelated to the amount of analyte in the medium.
2. Description of the Prior Art
Engasser and Horvath, Applied Biochem. Bioengineering, Vol. I, 127 (1976) Academic Press, report the kinetic and diffusion effects on the immobilization of enzymes. U.S. Pat. No. 3,817,837 describes a homogeneous enzyme immunoassay. U.S. Pat. No. 3,996,345 describes a homogeneous immunoassay employing two chromophores related by being a fluorescer and a quencher. Copending application Ser. No. 815,636, U.S. Pat. No. 4,160,645 filed July 14, 1977, describes a homogeneous immunoassay employing a nonenzymatic catalyst as a label, while copending application Ser. No. 815,632, U.S. Pat. No. 4,208,479, describes means for modulating signals in immunoassays. Copending application Ser. No. 906,514, U.S. Pat. No. 4,193,983, filed May 16, 1978, describes a labeled liquid discontinuous phase for use in immunoassays. Application Ser. No. 667,996, abandoned, filed Mar. 18, 1976, describes a homogeneous immunoassay employing as a label an enzyme substrate. See also U.S. Pat. No. 3,853,987, which discloses particles to which are conjugated radioactive and fluorescent labels and antibodies. See also U.S. Pat. No. 4,001,400. See also copending application Ser. No. 964,099, filed Nov. 24, 1978.